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. 2023 Feb 22;9(3):e13888. doi: 10.1016/j.heliyon.2023.e13888

Analysis of mitochondrial respiration and ATP synthase in frozen brain tissues

Pamela J Yao a,∗, Rachel Munk b, Myriam Gorospe b, Dimitrios Kapogiannis a,∗∗
PMCID: PMC9988573  PMID: 36895388

Abstract

Studying mitochondrial respiration capacity is essential for gaining insights into mitochondrial functions. In frozen tissue samples, however, our ability to study mitochondrial respiration is restricted by damage elicited to the inner mitochondrial membranes by freeze-thaw cycles. We developed an approach that combines multiple assays and is tailored towards assessing mitochondrial electron transport chain and ATP synthase in frozen tissues. Using small amounts of frozen tissue, we systematically analyzed the quantity as well as activity of both the electron transport chain complexes and ATP synthase in rat brains during postnatal development. We reveal a previously little-known pattern of increasing mitochondrial respiration capacity with brain development. In addition to providing proof-of-principle evidence that mitochondrial activity changes during brain development, our study details an approach that can be applicable to many other types of frozen cell or tissue samples.

Keywords: Mitochondrial respiration, Frozen samples, Brain, Development, Electron transport chain, ATP synthase

Highlights

  • •Workflow for measuring mitochondrial respiration in frozen tissues and cells.

  • •

    Uses small sample amount and commercially available reagents.

  • •

    Proof-of-principle experiment reveals respiration pattern in developing rat brain.

  • •

    Applicable to animal and human samples, bridging basic science and clinical studies.

1. Introduction

Mitochondria are known to have varied biological functions in cells [1,2]. Among these functions is the production of ATP via oxidative phosphorylation [3,4]. An adequate supply of ATP is critical to all cells including the neuron [[5], [6], [7]]. Thus, it is not surprising that, in neurons, disruption of mitochondrial respiration and ATP production leads to a wide range of neurological disorders such as Leigh syndrome [8], Fragile X syndrome [9], and Alzheimer's disease [10].

The oxidative phosphorylation system is composed of the electron transport chain, which contains four protein complexes (I–IV), and the ATP synthase (also known as complex V). The electron transport chain generates a proton gradient across the inner mitochondrial membrane that drives the ATP synthase to produce ATP [3,11]. The assessment of mitochondrial oxidative phosphorylation or respiration capacity in frozen samples has been hindered by the fact that cycles of freezing and thawing dismantles the electron transport chain and the ATP synthase. Therefore, information pertaining to mitochondrial respiration in frozen samples is often inferred from measurement of the quantity of specific proteins. In this study, we investigate mitochondrial respiration capacity in the rat brain during postnatal development using frozen tissue samples. We use a method designed for analysis of electron transport chain activity in frozen tissues [12,13]. In addition, we measure ATP synthase activity and quantify the levels of all five complexes. We uncover a developmental profile of mitochondrial respiration capacity that was previously unknown and present a practical approach for the functional analysis of mitochondria in any frozen tissue sample.

2. Results

2.1. Mitochondrial respiration in the developing rat brain

Investigations into cellular oxygen consumption – the readout of mitochondrial respiration – have been mostly carried out in live cells or fresh tissue samples. In frozen samples, freeze-thaw cycles can damage mitochondrial membranes resulting in disruption of the electron transport chain, as well as leakage of cytochrome c, both of which are needed for mitochondrial respiration. However, evidence indicates that electron transport chain protein complexes seem to remain intact and functional despite being subjected to the freeze-thaw process [14]. Based on this realization, a modified assay utilizing a combination of specific substrates and inhibitors, with the addition of cytochrome c, has successfully gauged the activity of the mitochondrial electron transport system in the form of oxygen consumption rate in frozen samples [12,13].

One important benefit of using frozen samples is that the experimental design can be widely expanded. Samples can be collected as they become available, divided into aliquots, and stored frozen until assays can be conducted. Another benefit of using frozen samples is that aliquots of the same sample can be used in new investigations addressing different questions. For example, in this study, we made use of the frozen rat brains that were collected while we were conducting previous brain development studies [15,16]. With these tissues, we aimed to analyze mitochondrial respiration in frozen samples, and to understand mitochondrial functions in the brain during postnatal development.

To determine the range of protein concentrations for the mitochondrial oxygen consumption rate (OCR) assay, we first tested different amounts of protein from tissue homogenates of frozen rat brains. Using total protein ranging from 2.5 to 30 μg, we observed robust complex II-mediated OCR elicited by succinate – a substrate for complex II (Supplementary Fig. 1A). Similarly, we observed that OCR was readily induced by TMPD (Tetramethyl-p-phenylenediamine dihydrochloride) – a complex IV substrate (Supplementary Fig. 1A). In both cases, the level of OCR positively correlated with the amount of protein (Supplementary Fig. 1A).

We next investigated mitochondrial respiration capacity in postnatal developing rat brains. We measured complex II- and complex IV-mediated OCR in the cortical brain samples of rats at different ages, from postnatal day 1 (p1) to young adulthood (p30). We used tissue homogenates that contained 20 μg of protein per sample.

From OCR traces (Fig. 1A; Supplementary Fig. 1B) and the averaged OCR values of 4–6 rats per age group (Fig. 1B), we found increased OCR from p1 to p21: with 61% increase in OCR by complex II, and 120% increase in OCR by complex IV (complex II: p1 = 211 ± 21.8 vs p21 = 340 ± 21.2, p = 0.004, n = 6 for p1, n = 4 for p21; complex IV: p1 = 88 ± 10.3 vs p21 = 194 ± 21, p = 0.001, n = 6 for p1, n = 4 for p21). From p21 to p30, both complex II- and complex IV-mediated OCR seemed to level off although they were still significantly higher than corresponding measures in p1 (complex II: p1 = 211 ± 21.8 vs p30 = 310 ± 16.3, p = 0.007, n = 5; complex IV: p1 = 88 ± 10.3 vs p30 = 160 ± 16.5, p = 0.004, n = 4).

Fig. 1.

Fig. 1

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Analysis of frozen brain tissues reveals an age-dependent increase of mitochondrial respiration in postnatal developing rats. (A) Example traces of oxygen consumption rate (OCR) of frozen brain tissue (homogenates, 20 μg of total protein) in rats from postnatal day 1 (p1) to p30. Each data point is an average of duplicate samples. Succinate/rot (rotenone), AA (antimycin A), TMPD/asc (Tetramethyl-p-phenylenediamine dihydrochloride/ascorbic acid), and sodium azide were injected sequentially (Materials and Methods). Succinate/rot-induced respiration represents complex II-mediated respiration; TMPD/asc-induced respiration represents complex IV-mediated respiration. (B) Quantification of (A). Shown in each bar is mean ± SEM. Each diamond represents data from one rat (n = 4–6 rats per age). *p < 0.05. p values were determined by unpaired Student's t-test. NS, not significant. (C) Example OCR traces of mitochondria (20 μg of protein) isolated from frozen brain tissues of p1 and p30 rats. (D) Quantification of (C). Shown in each bar is mean ± SEM. Each diamond represents data from one rat (n = 3 rats per age). *p < 0.05.

The observed age-dependent increase of OCR could simply reflect presumably increased mitochondrial mass (i.e. increased number and/or size of mitochondria) in the developing brain. To test this possibility, we assessed mitochondrial content in the brain tissue homogenates using a mitochondrial member-specific dye MitoTracker Deep Red (MTDR) [17]. MTDR signal intensity increased in a linear fashion in the range of 0.5–20 μg protein; but the signal became saturated at protein amounts of 30 μg or greater (Supplementary Fig. 1C). Therefore, we used homogenates containing 20 μg of protein, and compared mitochondrial content in the rat brain tissues; we did not observe significant differences between various age groups (Supplementary Fig. 1D). This suggests that, assessed by the mitochondrial member dye MTDR, total mitochondrial mass does not change significantly in postnatal developing rat brains.

To determine whether respiration capacity increases per mitochondrion during development, we isolated mitochondria from the frozen brain tissues then measured OCR. As expected, for the same amount of protein (20 μg protein), OCR levels were higher in isolated mitochondria than in tissue homogenates (compare Fig. 1C and D to A and B). More importantly, when we compared OCR between p1 and p30 mitochondria, we found that the averaged OCR value in p30 was significantly higher than in p1 (complex II: p1 = 407 ± 14.9 vs p30 = 502 ± 14.6, p = 0.01, n = 3 for both p1 and p30; complex IV: p1 = 207 ± 22.5 vs p30 = 323 ± 17.2, p = 0.015, n = 3 for both p1 and p30). These data suggest that the respiration capacity per mitochondrion increases in rat brains during postnatal development.

2.2. Mitochondrial ATP synthase activity in the developing rat brain

Although the mitochondrial OCR assay can gauge the activity of the electron transport chain complex I-IV in frozen samples, it does not measure ATP synthase, also known as complex V [11]. To further understand the mitochondrial oxidative phosphorylation process in the developing rat brain (Fig. 2A), we used a metabolic/colorimetric assay to measure ATP synthase activity and quantity. In this assay, mitochondrial ATP synthase is immunocaptured and its activity is quantified based on the conversion of NADH to NAD+ (Materials and Methods). A pilot experiment showed that the specific activity (the ratio of measured activity over quantity) of the ATP synthase correlated with the amounts of proteins in frozen brain homogenates (Supplementary Fig. 2).

Fig. 2.

Fig. 2

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ATP synthase activity in frozen brain tissues shows an age-dependent increase in postnatal developing rats. (A) Schematic of the electron transport chain (complex I to IV) and ATP synthase (complex V). Drawing created with BioRender. (B) ATP synthase activity in brains of postnatal rats from p1 to p30. Shown in each bar is mean ± SEM. Each diamond represents data from one rat (n = 4 rats per age). *p < 0.05 (comparisons to p1, as reference group). p values were determined by unpaired Student's t-test.

In the developing rat brain, we found that, similar to the pattern observed for complex II- and complex IV-mediated OCR, ATP synthase specific activity also increased with age: its level nearly doubled in p14 as compared to that at p1 (p14 = 198 ± 18.3 vs p1 = 100, p = 0.002, n = 4), and stayed at a high level in p21 (p21 = 199 ± 30.8 vs p1 = 100, p = 0.02, n = 4) (Fig. 2B). In the p30 brain, ATP synthase activity leveled off, although it was still higher than that in p1 (p30 = 150 ± 14 vs p1 = 100, p = 0.01, n = 4). These data show that the activity of the oxidative phosphorylation system increases as the brain grows.

2.3. Mitochondrial protein levels in developing rat brain

We next examined the levels of the mitochondrial complexes I–IV and ATP synthase (complex V) using immunoblotting. We compared the levels of five complexes to the levels of mitochondrial proteins that are not directly involved in mitochondrial respiration, such as TOMM20 (translocase of outer membrane) and VDAC (voltage-dependent anion channel). In addition, we measured synaptophysin, a synaptic vesicle protein [18], as a surrogate for developing neurons and synapses, and actin as a control for protein loading.

We used an antibody-cocktail to detect all five complexes simultaneously. Complex V signal was robust as early as p1 and continued to increase slightly with age; complex III signal was readily visible at p1 and increased steadily onward; complex I and II were barely detectable at p1 but increased noticeably from p14 to p20 (Fig. 3A; Supplementary Figs. 3 and 4). We normalized the proteins bands for the five complexes to two controls, actin for total cellular proteins or TOMM20 for mitochondrial proteins. As illustrated in Fig. 3B, complex I and II showed nearly identical patterns of developmental age-dependent increase, regardless of whether the protein bands were normalized to actin or TOMM20. For complex III and V, the overall upward temporal trends were similar for both actin and TOMM20 normalization, at most age points (Fig. 3B). For complex IV, the corresponding protein band was faint on some blots (for example, Fig. 3A; Supplementary Fig. 4A); therefore, we detected it with another antibody (coxIV) and the resulting blot showed noticeably and significantly higher levels of complex IV starting from p14 (Fig. 3C and D; Supplementary Figs. 4B and D).

Fig. 3.

Fig. 3

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Levels of mitochondrial respiration chain proteins and ATP synthase in postnatal rat brains. (A) An example blot showing mitochondrial respiration chain complexes I to IV and ATP synthase (complex V) in the cortical tissues of rats age from p1 to p30. See Supplementary Fig. 3 for uncropped version of the blot, and Supplementary Figs. 4A and C, for blots of additional rat samples. (B) The level of each protein was normalized to either the level of corresponding actin (upper panel) or TOMM20 (lower panel). Shown in each bar is mean ± SEM. Each diamond represents data from one rat (n = 3 rats per age). *p < 0.05 (comparisons to p1, as reference group, gray dashed line). p values were determined by unpaired Student's t-test. NS, not significant. (C) Example blots of mitochondrial respiration chain complex IV (coxIV), mitochondrial outer membrane protein TOMM20 and VDAC, synaptic protein synaptophysin (SYP), and actin. See Supplementary Fig. 3 for uncropped version of the blots, and Supplementary Figs. 4B and D for blots of additional rat samples. (D) The level of each protein was normalized to the level of corresponding actin. Similar to (B), shown in each bar is the mean value from 3 rats. Each diamond represents data from one rat. *p < 0.05 (comparisons to p1, as reference group, gray dashed line). p values were determined by unpaired Student's t-test. NS, not significant.

TOMM20 levels remained relatively constant from p1 to p30 (Fig. 3C) in two of the three rats examined (Supplementary Fig. 4). Averaging the values from blots of all three rats revealed no significant differences between different ages (Fig. 3D). VDAC level showed a small statistically significant increase at p30 but not at earlier ages (Fig. 3D). Synaptophysin (SYP), on the other hand, began to elevate at p2 then increased drastically from p14 onward (Fig. 3D; Supplementary Fig. 4).

Together, these data indicate that the activity as well as quantities of mitochondrial electron transport chain complexes and ATP synthase in the brain increase during postnatal development.

3. Discussion

Our study had two objectives. First, we sought to analyze mitochondrial function, specifically mitochondrial respiration, in frozen samples. We designed a fairly simple experimental strategy by using a combination of three methods: OCR measurement for electron transport chain activity [12,13], an immuno/colorimetric assay for ATP synthase activity (commercially available), and immunoblotting with an antibody-cocktail for simultaneously evaluating quantities of electron transport chain complexes I-IV and ATP synthase (also commercially available).

The second objective of our study was to investigate mitochondrial respiration in the brain during development, particularly during postnatal development, an age when active axon and dendrite growth and synaptogenesis take place. In addition to serving as a proof-of-principle study, we hoped to elucidate the pattern of mitochondrial respiration in the developing brain, a piece of information that was previously unknown.

It is widely recognized that properly functioning mitochondria are integral to normal brain development [6,[19], [20], [21]]. A new study has shown that different pace of neuronal development among species is attributable to differences in mitochondrial metabolism, especially oxidative phosphorylation [22]. Our findings provide a clearer view of mitochondrial respiration capacity in the developing brain. A rising mitochondrial respiration capacity that is in parallel with the rising level of synaptophysin during postnatal development implies that a high level of energy supply must accompany synapse formation. This fundamental knowledge can lead to further understanding of malfunctioning mitochondria in abnormal brain development.

Regarding TOMM20 and VDAC, it is noteworthy that their levels are already high in the brain at birth (p1), noticeably higher than most electron transport chain proteins (Fig. 3; Supplementary Fig. 4). This suggests an underlying complexity that should be considered when studying differing mitochondrial functions. For future studies, it would be important to characterize the mitochondrial respiration capacity as well as the expression levels of mitochondrial proteins, including TOMM20 and VDAC, in the prenatal brain. It would also be important to analyze the mitochondrial activity in the developing brain comparing frozen tissues samples to fresh samples.

Our experimental approach does not require intensive resources. All the reagents are available commercially (see Materials and Methods). As for the frozen samples, less than a half milligram of tissues from each sample is sufficient for the mitochondrial OCR assay, ATP synthase and immunoblots. It should be noted that we used frozen sample aliquots of the developing rat brain tissues that were used previously in other developing brain studies [15,16], illustrating an example of enabling a new investigation by using prior samples collected for different studies. In the case of clinical investigations where frozen samples are usually the only option, this approach will be a powerful and practical way to characterize mitochondrial functions in clinical samples, thus, broadening our understanding of the pathogenic roles of mitochondria play in various diseases.

4. Materials and Methods

4.1. Frozen rat brain tissues

All animal procedures were approved by the NIA Animal Care and Use Committee. Postnatal Sprague-Dawley rats (postnatal day 1 to day 30) of either sex were used as the source of brain cortical tissues. The samples were collected while conducting previous studies and had been stored at −80 °C for 5 years [15,16]. The cortical issue of each rat was cut into multiple small pieces (∼0.5 mg per piece). Using a pair of pre-cooled forceps, each piece of tissue was placed in a 2 ml cryovial tube (empty but pre-labeled); after closing the lid, the tube was immediately placed in a thermoconductive tube rack that were sitting on crushed dry ice. The flash frozen samples were then transferred to a −80 °C and stored until tissue processing.

4.2. Oxygen consumption rate (OCR) measurement

We measured OCR following a recently established protocol designed for analyzing mitochondrial oxidative function in frozen tissues [12,13]. After rapidly thawing on slush ice-containing water, rat brain cortical tissues were homogenized in cold MAS assay buffer using a pestle motor mixer (Argos) in 1.5-ml microtubes. MAS buffer: 70 mM sucrose, 220 mM mannitol, 5 mM KH2PO4, 5 mM MgCl2, 1 mM EGTA, 2 mM HEPES, pH 7.4. The homogenates were centrifuged at 1000×g for 5 min at 4 °C, and the supernatants were collected. For some samples, the supernatants were further processed for mitochondrial isolation following the protocol by Osto et al., [12,13].

Protein concentration of the supernatants or isolated mitochondria was then determined using Pierce BCA protein assay kit (Thermo Fisher Scientific).

The samples containing 20 μg of total protein or mitochondrial protein in 20 μl of the MAS buffer were loaded to each well of a Seahorse XF24 cell culture plate (Agilent). After centrifuging at 2000×g for 5 min at 4 °C, 130 μl of MAS buffer containing cytochrome c (10 μg/ml) and alamethicin (10 μg/ml) was added to each well.

The sensor cartridge plate (Agilent) was loaded with substrates and inhibitors (50 μl per port) as follows: port A, succinate + rotenone (5 mM + 2 μM); port B, antimycin A (4 μM); port C, TMPD + ascorbic acid (0.5 mM + 1 mM); port D, sodium azide (50 mM). These conditions allow for the measurement of complex II- and IV-mediated maximal respiratory capacity [12,13].

The samples were run in duplicates in each assay. Cortical tissues from 4 to 6 rats per age group were assayed.

4.3. ATP synthase measurement

We measured ATP synthase activity and quantity in the rat cortical tissues using a commercial assay (abcam; ab109716). ATP synthase is immunocaptured in the wells of an assay plate and the conversion of ATP to ADP by ATP synthase is coupled to the conversion of NADH to NAD+, with the formation of the latter being monitored as absorbance change. This assay is intended for measuring both the activity and quantity of ATP synthase. The ratio of the two measurements represents the specific activity of ATP synthase.

We followed the manufacturer's instructions with minor modifications. After rapidly thawing on slush ice-containing water, rat brain cortical tissues were homogenized in the assay buffer (solution 1) with 10% detergent (both provided in the assay kit). The homogenates were centrifuged at 10,000×g for 10 min at 4 °C, the supernatants were collected, and the protein concentration was determined using the BCA assay. The samples containing 20 μg of total protein in 50 μl of the solution 1 assay buffer were added to the wells of the assay plate. The plate was incubated overnight with gentle rocking at 4 °C followed by the measurement of ATP synthase activity, quantity and specific activity (ratio of activity to quantity), per the manufacturer's instruction. The samples were run in duplicates in each assay; 4 rats per age were assayed.

4.4. Mitochondrial content measurement

We measured mitochondrial content following the protocol by Osto et al. [12,13], with minor modifications. After homogenizing rat brain cortical tissues in cold MAS assay buffer followed by protein concentration measurements with the BCA protein assay, we added 50 μl of protein homogenates containing 20 μg protein to wells of a clear flat-bottom black 96-well plate, followed by the addition of MTDR (MitoTracker Deep Red) at 1 μM final concentration, in 100 μl total volume per well. The plate was incubated at 37 °C for 10 min, then centrifuged at 2000×g, 4 °C for 5 min. We carefully removed the supernatant containing unbound MTDR dye, and then washed the wells once by gently adding then removing 100 μl MAS buffer. After adding another 100 μl of MAS buffer, we measured fluorescence using a plate reader. All samples were run in quadruplicates.

4.5. Mitochondrial protein level measurement – immunoblot analysis

We followed standard immunoblotting procedures. After rapidly thawing on ice slush ice-containing water, rat brain cortical tissues were homogenized in RIPA bufer (Thermo Fisher Scientific) containing Halt protease and phosphatase inhibitors (Thermo Fisher Scientific). Following centrifugation at 10,000×g for 10 min at 4 °C, the supernatants were collected and the protein concentration was determined using the BCA assay. The samples (20 μg of total protein per well) were resolved on 4–12% Bis-Tris NuPAGE gels and transferred to nitrocellulose membranes (0.2-μm pore size). The membranes were then incubated with primary antibodies at 4 °C overnight followed by species-appropriate, horseradish peroxidase (HRP)-linked secondary antibodies at RT for 1 h. The bound antibodies were visualized by the enhanced chemiluminescence method (Pierce) on reflection autoradiography films.

The following antibodies were used: total OXPHOS rodent WB antibody cocktail (#ab110413, abcam); coxIV antibody (#11967, Cell Signaling Technology); TOMM20 antibody (#186734, abcam); VDAC antibody (#4661, Cell Signaling Technology); synaptophysin antibody (#S5678, Millipore Sigma); actin antibody (#A5441, Millipore Sigma), and HRP-linked IgG from rabbit and mouse (7074S and 7076S, Cell Signaling Technology).

4.6. Data analysis

Statistical analysis was performed using KaleidaGraph (Synergy Software). Groups were compared using unpaired Student's t-test. The values represent the mean ± SEM from 3 to 6 independent rat samples.

Author contribution statement

Pamela J Yao: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Rachel Munk: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Myriam Gorospe; Dimitrios Kapogiannis: Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data included in article/supp. material/referenced in article.

Declaration of interest's statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This study was supported by the Intramural Research Programs of the National Institutes of Health, National Institute on Aging.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2023.e13888.

Contributor Information

Pamela J. Yao, Email: Pamela.Yao@nih.gov.

Dimitrios Kapogiannis, Email: kapogiannisd@mail.nih.gov.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

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mmc1.docx (22.6MB, docx)

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